Method for detecting presence or absence of heteroploidy of fetal chromosome

Abstract

A method for detecting the presence or absence of heteroploidy of fetal chromosomes includes: obtaining a primary amplification products by amplifying chromosomal DNA obtained from a biological sample collected from a pregnant mother; performing multiplex amplification of a plurality of target regions to obtain a secondary amplification product using the primary amplification product as a template; adding a label to both terminals of the secondary amplification product to obtain the labeled secondary amplification product; performing amplification using a primer pair annealing to the label using the labeled secondary amplification product as a template to obtain a tertiary amplification product; and determining the amplification amount and base sequences of the plurality of target regions from the tertiary amplification product. The primary amplification amplifies the total amount of DNA by 6000 to 30000 times, and the secondary amplification step amplifies the total amount of DNA by 3 to 150 times.

What is claimed is: 
     
         1 . A method for detecting the presence or absence of heteroploidy of fetal chromosomes, the method comprising:
 a primary amplification step of obtaining a primary amplification products by amplifying chromosomal DNA obtained from a biological sample collected from a pregnant mother;   a secondary amplification step of performing multiplex amplification of a plurality of target regions using a plurality of primer pairs to obtain a secondary amplification product using the primary amplification product as a template;   a first addition step of adding a first label, which is oligonucleotide, to both terminals of the secondary amplification product to obtain the labeled secondary amplification product;   a tertiary amplification step of performing amplification using a primer pair annealing to the first label using the labeled secondary amplification product as a template to obtain a tertiary amplification product; and   a sequence analysis step of determining the amplification amount and base sequences of the plurality of target regions from the tertiary amplification product,   wherein the primary amplification step is a step of amplifying the total amount of DNA by 6000 times to 30000 times, and   wherein the secondary amplification step is a step of amplifying the total amount of DNA by 3 times to 150 times.   
     
     
         2 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the tertiary amplification step is a step of amplifying the total amount of DNA by 6 times to 24 times.   
     
     
         3 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the sequence analysis step is performed using a next-generation sequencer.   
     
     
         4 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 , the method further comprising:
 a second addition step of adding a second label, which is oligonucleotide, to both terminals of the tertiary amplification product to obtain the labeled tertiary amplification product.   
     
     
         5 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 4 ,
 wherein the primer used in the tertiary amplification step has a base sequence of the second label at the 5′-terminal, and   wherein the second label is added to both terminals of the tertiary amplification product and the labeled tertiary amplification product is obtained by performing the tertiary amplification step which is performed together with the second addition step in this manner.   
     
     
         6 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the primer used in the secondary amplification step has a base sequence of the first label at the 5′-terminal, and   wherein the first label is added to both terminals of the secondary amplification product and the labeled secondary amplification product is obtained by performing the secondary amplification step which is performed together with the first addition step in this manner.   
     
     
         7 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the primary amplification step is a step of performing whole genome amplification from the chromosomal DNA.   
     
     
         8 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the chromosomal DNA is chromosomal DNA obtained from one cell, and   wherein the primary amplification step is a step of performing whole genome amplification from one cell.   
     
     
         9 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 ,
 wherein the biological sample is blood, and   wherein the chromosomal DNA is chromosomal DNA obtained from a cell in blood.   
     
     
         10 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 9 ,
 wherein the cell in blood is a nucleated red blood cell.   
     
     
         11 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 9 ,
 wherein the cell in blood is a cell isolated through density gradient centrifugation and image analysis.   
     
     
         12 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 , wherein the biological sample comprises fetus-derived cells. 
     
     
         13 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 , further comprising a step of determining a proportion between the amplification amounts of the plurality of target regions in the tertiary amplification product. 
     
     
         14 . The method for detecting the presence or absence of heteroploidy of fetal chromosomes according to  claim 1 , wherein the sequence analysis step comprises determining an amount of a mother-derived tertiary amplification product as well as an amount of a fetus-derived tertiary amplification product.